Synthesis and biological evaluation of novel pyrazole compounds

Article abstract of DOI:10.1016/j.bmc.2010.06.018

A novel dipyrazole ethandiamide compound and acid chloride of pyrazolo[3,4-d]pyrimidine 4(5H)-one were prepared and reacted with a number of nucleophiles. The resultant novel compounds were tested in several in vitro and in vivo assays. Three compounds inhibited the secretion of neurotoxins by human THP-1 monocytic cells at concentrations that were not toxic to these cells. They also partially inhibited both cyclooxygenase-1 and -2 isoforms. In animal studies, two compounds were notable for their anti-inflammatory activity that was comparable to that of the clinically available cyclooxygenase-2 inhibitor celecoxib. Modeling studies by using the molecular operating environment module showed comparable docking scores for the two enantiomers docked in the active site of cyclooxygenase-2.

Full text of DOI:10.1016/j.bmc.2010.06.018

Bioorganic & Medicinal Chemistry 18 (2010) 5685–5696
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel pyrazole compounds
Amal M. Youssef a,b, Edward G. Neeland a, Erika B. Villanueva c, M. Sydney White c,
a,
Ibrahim M. El-Ashmawy d, Brian Patrick e, Andis Klegeris c, , Alaa S. Abd-El-Aziz
*
*
a Department of Chemistry, University of British Columbia Okanagan, Kelowna, BC, Canada
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
c Department of Biology, University of British Columbia Okanagan, Kelowna, BC, Canada
d Department of Pharmacology, Faculty of Veterinary Medicine, University of Alexandria, Alexandria, Egypt
e X-ray Facility, University of British Columbia Vancouver, BC, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel dipyrazole ethandiamide compound and acid chloride of pyrazolo[3,4-d]pyrimidine 4(5H)-one
were prepared and reacted with a number of nucleophiles. The resultant novel compounds were tested
in several in vitro and in vivo assays. Three compounds inhibited the secretion of neurotoxins by human
THP-1 monocytic cells at concentrations that were not toxic to these cells. They also partially inhibited
both cyclooxygenase-1 and -2 isoforms. In animal studies, two compounds were notable for their anti-
inflammatory activity that was comparable to that of the clinically available cyclooxygenase-2 inhibitor
celecoxib. Modeling studies by using the molecular operating environment module showed comparable
docking scores for the two enantiomers docked in the active site of cyclooxygenase-2.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 28 April 2010
Revised 4 June 2010
Accepted 5 June 2010
Available online 12 June 2010
Keywords:
Pyrazoles
Anti-inflammatory drugs
Neuroprotection
1. Introduction
for the treatment of pain and inflammation. Celecoxib (aka: Celeb-
rexÒ, CelebraÒ, NiflamÒ, and OnsenalÒ), a COX-2-selective NSAID is
Pyrazole and pyrazolopyrimidine compounds have been re-
ported as therapeutic agents for a diverse number of applications.
Pyrazole derivatives have been proposed as potential treatments
for neurodegenerative disorders like Alzheimer’s disease, Parkin-
son’s disease, and bovine spongiform encephalopathy.1 Pyrazoles
are also active antitumour,2 anticonvulsant,3 and antimicrobial
agents.4 They are used for pain relief5 and have shown success in
the inhibition of the human immunodeficiency virus.6 Pyrazole
derivatives are insecticides7 and are used in dyeing.8
a pyrazole derivative. Celecoxib has relatively few gastrointestinal
(GI) side effects but its long-term use is associated with cardiovas-
cular injury, which limits its clinical applications.18 Therefore,
there is a need for the development of novel drugs with better
safety profiles that could be used long-term to relieve chronic
inflammatory conditions. Our starting point was synthesizing
new derivatives of pyrazoles and investigating how structural
changes affected their anti-inflammatory and neuroprotective
properties.
Pyrazolopyrimidine compounds are equally widespread in
diversity. They may act as Trk and c-Src inhibitors,9 C–C chemokine
receptor type 1 (CCR1) and purine antagonists,10 HIV reverse trans-
criptase inhibitors11 as well as showing activity as antitumour/
antileukemia,12 and herbicidal/fungicidal13 agents. Like the pyra-
zoles, pyrazolopyrimidines are used in the dyeing industry.14 Pyr-
azolopyrimidines are also used to treat insomnia.15
We were most interested in the anti-inflammatory properties of
pyrazoles and pyrazolo[3,4-d]pyrimidines as potential inhibitors of
cyclooxygenase (COX)16 and p38a17 isoenzymes. Non-steroidal
anti-inflammatory drugs (NSAIDs), which are known to inhibit
COX enzymes, are currently used as important therapeutic agents
2. Results and discussion
2.1. Chemistry
Popular methods for making a pyrazole ring system react a
hydrazine moiety with
centers. These centers are represented by
bonyl,19 ,b-unsaturated nitrile,12c b-ketocarbonyl,3,5a or b-nitrilo-
carbonyl functionalities2a,9a,10b,20 (Scheme 1).
Other approaches to pyrazoles react a substituted hydrazine
with an
-chlorocarbonyl group.21 Once formed, the pyrazole rings
a
molecule possessing b-electrophilic
a
,b-unsaturated car-
a
a
can be further elaborated to pyrazolopyrimidines using formamide
or urea,10b,22 chromenones,23 or base catalyzed cyclization.12d,24,25
We followed a modified method of Cheng10b whereby phen-
ylhydrazine and ethoxymethylenemalonitrile reacted to produce
* Corresponding authors. Tel.: +1 250 807 9557; fax: +1 250 807 8005 (A.K.); tel.:
+1 250 807 9139; fax: +1 250 807 9005 (A.S.A.).
E-mail addresses: andis.klegeris@ubc.ca (A. Klegeris), alaa.abd-el-aziz@ubc.ca
(A.S. Abd-El-Aziz).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.018

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A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
with excess NH4OH only produced starting material. The reluc-
R1
R1
R2
R2
tance of the second pyrazole ring to leave may be attributed to
its carbonyl group being inductively stabilized by the newly intro-
duced and more electron-donating amino substituent.
The formation and reactivity of the dipyrazole ethandiamide 3
suggested a revised synthetic route to make the originally desired
pyrazolo[3,4-d]pyrimidine 2. Very slow addition of pyrazole 1 to an
excess of oxalyl chloride produced the acid chloride of pyrazol-
o[3,4-d]pyrimidine which was immediately reacted with methanol
to yield the methyl ester 2 (Fig. 3).
δ
H N
N
δ
2
R3
R3
NH
Z
N
Z
Scheme 1. Formation of the pyrazole ring.
This series of unique compounds, dipyrazole ethandiamide 3,
pyrazole ethandiamides 4a–f, and the methyl pyrazolo[3,4-
d]pyrimidine ester 2, were subsequently tested for potential anti-
inflammatory and neuroprotective properties.
2-amino-3-nitrilo-1-phenylpyrazole 1. Reaction of 1 with oxalyl
chloride followed by nucleophilic attack was expected to yield
substituted pyrazolo[3,4-d]pyrimidines 2 (Scheme 2).
In our hands, this reaction gave an 85% yield of a crystalline par-
ent compound, which cleanly reacted with a variety of amine
nucleophiles. However, the spectral analysis showed that the par-
ent compound had nine unique carbon atoms, possessed a nitrile
functionality and an independent phenyl substituted pyrazole ring.
This suggested that the pyrazole amine 1 had undergone a bimo-
lecular addition with the oxalyl chloride to yield the dipyrazole
ethandiamide compound 3 (Scheme 3).
Heterocyclic ethandiamide derivatives like this are infrequently
reported but not unknown.1a,1b,4d,26 Suitable crystals of 3 were ana-
lyzed by X-ray crystallography and the Oakridge Thermal Ellipsoid
Plot (ORTEP) confirmed the dipyrazole ethandiamide structure
(Fig. 1).
With the structure of the parent compound 3 in hand, we
turned our attention to the products which had incorporated the
amine nucleophiles. The amine adducts did not exist as tautomeric
Schiff bases27 of compound 3. Two dimensional NMR data from
correlation spectroscopy (COSY-45) and heteronuclear multiple
quantum coherence (HMQC) identified two imine (NH) hydrogens
of which one NH showed J-coupling with the substituent R group
and the other NH was uncoupled. This pattern is inconsistent with
the Schiff base structure. A nuclear Overhauser enhancement
(NOE) was also observed between the NH adjacent to the pyrazole
ring and the ortho phenyl hydrogen. Furthermore, heteronuclear
multiple bond coherence (HMBC) correlations were observed be-
tween one NH and carbonyl carbon atom as well as between the
methine (CH) of the amine substituent and the other carbonyl car-
bon atom (Fig. 2).
2.2. In vitro assays
The above compounds were initially tested in several in vitro
assays to assess their cytotoxic, anti-inflammatory, and anti-neu-
rotoxic properties. Figures 4–7 show data obtained by using the
lead compound 3, while Table 1 summarizes data obtained in the
same assays by using all six of the newly synthesized compounds
as well as a standard anti-inflammatory drug celecoxib.
2.2.1. Monocytic cell viability and chemokine secretion
First, human monocytic THP-1 cells were pre-incubated with
experimental drugs or the vehicle solution dimethyl sulfoxide
(DMSO) for 15 min before their stimulation with a combination
of lipopolysaccharide (LPS) and interferon (IFN)-c. Viability of
THP-1 cells and secretion of a pro-inflammatory chemokine mono-
cyte chemoattractant protein-1 (MCP-1) by these cells were as-
sessed 24 h later. Figure 4 shows that at 500 lM, compound 3
caused a significant reduction in the percentage of viable THP-1
cells (Fig. 4A) and increased lactate dehydrogenase (LDH) concen-
tration in the supernatants, which indicates increased cellular lysis
(Fig. 4B). Only the highest concentration of compound 3 studied
(500 lM) caused a reduction in MCP-1 levels after the 24 h incuba-
tion period (Fig. 5), which was most likely caused by the direct tox-
icity to THP-1 cells. Table 1 illustrates that the inhibitory effects of
compound 2 and celecoxib on MCP-1 secretion were similarly due
to their direct toxicity. Meanwhile the IC50 for compound 4d on
MCP-1 secretion (Table 1) was lower than the EC50 for its toxic ef-
fect, which may indicate a selective anti-inflammatory activity of
this compound.
These data, taken together, were more suggestive of a monopy-
razolo diamide 4 resulting from a pyrazole displacement by the
amine nucleophile (Scheme 4).
To avoid going through the intermediate compound 3, the
structure of 4a was confirmed via an independent synthesis in this
order: oxalyl chloride, phenethylamine, and pyrazole 1. Curiously,
no evidence of both pyrazole rings being displaced in these reac-
tions was observed. Despite reacting 3 with 10 equiv of phenethyl-
amine and substantially increasing the reaction time, compound
4a was still isolated resulting from the loss of only one pyrazole
ring. Further attempts to displace the second pyrazole ring in 4a
2.2.2. Anti-neurotoxic effects
Figure 6 illustrates the anti-neurotoxic effect of compound 3,
which was assessed by measuring the viability of human neuronal
SH-SY5Y cells after their exposure to THP-1 cell supernatants for
72 h. In previous studies,28 supernatants from unstimulated THP-1
cells did not cause a reduction of SH-SY5Y cell viability and LDH
levels were comparable to those obtained after incubation of SH-
SY5Y cells in growth medium only (data not shown). Transfer of
O
EtO
CN
CN
NH
b,c
a
H2N
N
N
Nu
NH
N
N
N
N
NH2
O
Ph
Ph
Ph
1
2
Scheme 2. Reagents and condition: (a) EtOH, reflux; (b) (COCl)2; (c) Nuꢀ.

A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
5687
NC
CN
CN
O
(COCl)2
N
N
N
H
N
a
N
N
N
NH2
N
H
Ph
Ph
Ph
O
1
3
Scheme 3. Formation of the diadduct with oxalyl chloride. Reagent and condition: (a) THF, rt.
N
N
N
O
NH
N
NH
O
N
N
H
Figure 1. ORTEP plot and structure of 3; 50% probability thermal ellipsoids; selected bond lengths (Å) and angles (°): C(15)–C(16) 1.5308 (16), C(15)–O(1) 1.2134 (14), C(15)–
N(14) 1.3539 (15), C(5)–N(14) 1.3942 (15), C(5)–C(4) 1.3872 (17), C(5)–N(1) 1.3536 (16), C(4)–C(12) 1.4235 (19), C(4)–C(3) 1.4057 (18), C(12)–N(13) 1.1492 (19), C(3)–N(2)
1.3198 (18), N(2)–N(1) 1.3737 (14), N(1)–C(6) 1.4352 (16); N(2)–C(3)–C(4) 112.01 (11), C(5)–C(4)–C(3) 104.45 (11), C(5)–C(4)–C(12) 127.03 (12), C(3)–C(4)–C(12) 128.52
(12), N(1)–C(5)–N(14) 124.85 (11), C(15)–N(14)–C(5) 123.65 (10), N(14)–C(15)–C(16) 111.90 (10), N(13)–C(12)–C(4) 178.54 (14), O(1)–C(15)–N(14) 125.95 (11).
CN
CN
stimulation reduced the neurotoxicity of THP-1 cell secretions. At
the highest concentration of 500 M, there was pronounced neuro-
nal death, again, most likely caused by direct toxic effect of com-
pound 3 that was transferred with supernatants to the neuronal
cells. We confirmed that compound 3 was directly toxic to the SH-
H
N
O
N
l
N
N
N
N
H
N
H
H
O
H
SY5Y cells by applying it at 5–500 lM to SH-SY5Y cultures and mea-
suring cell viability 72 h later. According to the MTT assay, com-
pound 3 caused an 86% and 23% reduction in cell viability at 500
HMBC
NOE
and 100 lM, respectively (data not shown).
Figure 2. Diagnostic NOE and HMBC correlations in compound 4.
With the completion of the pilot study, six of the newly synthe-
sized compounds plus the reference anti-inflammatory drug cele-
coxib were tested in the assays described above and the results
are summarized in Table 1.
Both MTT and LDH assays were performed to assess the cyto-
toxic and anti-neurotoxic effects of compounds and the corre-
supernatants from THP-1 cells that had been stimulated with
LPS + IFN- caused a significant reduction in SH-SY5Y cell viability
and increase in LDH content in cell supernatants. Addition of
c
10–100
lM of compound 3 to THP-1 cells 15 min prior to their
NC
CN
CN
O
O
N
N
N
H2N-R
H
N
H
N
R
N
N
N
N
H
a
N
H
Ph
Ph
Ph
O
O
3
4a: R= -CH2CH2Ph
4b: R= -CH2CH2CH2morpholino
4c: R= -cyclohexyl
4d: R= -( )-methylbenzyl
4e: R= -(+)-methylbenzyl
4f: R= -(-)-methylbenzyl
Scheme 4. Pyrazole diamide derivatives 4. Reagent and condition: (a) THF, reflux.

5688
A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
O
NH
O
N
N
N
O
Figure 3. ORTEP plot and structure of 2; 50% probability thermal ellipsoids; selected bond lengths (Å) and angles (°): N(1)–N(2) 1.3765 (13), N(1)–C(12) 1.4311 (14), N(1)–
C(9) 1.3662 (14), N(2)–C(3) 1.3176 (15), C(3)–C(8) 1.4044 (16), C(8)–C(9) 1.3891 (15), C(8)–C(4) 1.4319 (16), C(4)–O(1) 1.2297 (15), C(4)–N(5) 1.3912 (15), N(5)–C(6) 1.3657
(14), C(6)–N(7) 1.3015 (14), C(6)–C(10) 1.45103 (15), N(7)–C(9) 1.3686 (14); N(2)–C(3)–C(8) 110.91 (11), O(1)–C(4)–N(5) 121.82 (11), O(1)–C(4)–C(8) 126.61 (11), N(5)–
C(4)–C(8) 111.57 (10), C(9)–C(8)–C(3) 105.66 (10), N(1)–C(9)–N(7) 127.00 (10), N(7)–C(6)–N(5) 126.00 (10), N(5)–C(6)–C(10) 113.48 (10), N(7)–C(9)–C(8) 126.43 (10).
Figure 5. Effect of compound
3 on the secretion of the pro-inflammatory
chemokine MCP-1 by human monocytic THP-1 cells. Cells were treated as described
in the legend of Figure 4. MCP-1 concentration in cell-free supernatants was
measured by enzyme linked immunoabsorbent assay (ELISA). Data (means SEM)
from eight independent experiments are presented. The concentration-dependent
effect of compound 3 was assessed by randomized block design ANOVA.
monocytic cell neurotoxicity at significantly lower concentrations.
The difference between the toxic and anti-neurotoxic effect was
lowest for compound 2 (40
ference for compound 3 (100
vs 5 M); and the highest difference was for compound 4d, which
was toxic to THP-1 cells with an EC50 of 500 M, while its anti-neu-
rotoxic effects had an IC50 of 14 M and 6 M according to the MTT
l
M vs 20
l
M); there was a 10-fold dif-
l
M vs 10
lM) and celecoxib (50
lM
l
Figure 4. Direct toxicity of compound 3 on stimulated human monocytic THP-1
cells. Cells were seeded into 24-well plates at a concentration of 4.5 ꢁ 105 cells per
well in 0.9 mL of DMEM-F12 medium containing 5% fetal bovine serum (FBS). They
l
l
l
were pretreated with various concentrations (
abscissa) for 15 min before stimulation with lipopolysaccharide (LPS) (0.5 mg mLꢀ1
and IFN-
(150 U mLꢀ1). After 24 h incubation, the THP-1 cell viability was assessed
lM) of inhibitors (shown on the
and LDH assays, respectively. Compounds 2, 3, and 4d were there-
fore selected for further studies.
)
c
by the MTT assay (A) and by measuring the LDH activity in the supernatants (B).
Data (means SEM) from 11 independent experiments are presented. The concen-
tration-dependent effects of compound 3 were assessed by randomized block
design ANOVA; F and P values obtained for each of the drug treatments are
presented in the figure.
2.2.3. COX enzymatic activity
A COX inhibitor screening assay showed that all three com-
pounds partially inhibited both COX isoforms, while celecoxib, as
expected, was active mainly against the COX-2 enzyme (Table 2).
It is important to note that this assay was used as a general inhib-
itor screening tool; more accurate measurements, such as O2 up-
take assay, will be needed in order to obtain accurate estimates
of IC50 for the newly synthesized drugs.29
sponding EC50 and IC50 values were estimated. The lower concen-
tration value is presented in the table where the values obtained
by two assays differed. Two of the compounds (4b and 4c) showed
no significant effects in the three assays used. Compound 4a was
2.2.4. Neuroprotective effects
highly toxic to THP-1 cells with EC50 = 10
was not tested in other assays. Compounds 2, 3, 4d, and celecoxib,
although toxic to THP-1 cells at high concentrations, inhibited
l
M and therefore it
The direct neuroprotective effects of compounds 2, 3, and 4d
were also assessed. Addition of compound 3 directly to SH-SY5Y
cells at the time of transfer of supernatants from stimulated

A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
5689
Table 1
Comparative in vitro biological activity of pyrazole derivatives
Compound Concentration THP-1
THP-1
Anti-neurotoxic
(l
M) cytotoxicity secretion
effect IC50 (lM)
EC50
(
lM)
of MCP-1
IC50 (lM)
2
3
4a
4b
4c
4d
Celecoxib
0.5–500
5–500
0.5–500
5–500
5–500
1–1000
5–100
40a
100
10
>500
80
100
250
Not tested
>500
>500
150
20
10
Not tested
100
250
6
500
50
70
5
a
Bold values indicate assays where the concentration-dependent effects of the
compounds were statistically significant (P < 0.05, randomized block design
ANOVA). Cytotoxicity, inhibition of MCP-1 secretion and anti-neurotoxic activity
(inhibition of THP-1 toxicity towards SH-SY5Y cells) were assessed as described for
compound 3 in Figures 4–6, respectively.
Table 2
Inhibition of cyclooxygenase (COX-1 and COX-2) enzymatic activity
Compound
Concentration (number
of experiments)
COX-1 activity
(% inhibition)
COX-2 activity
(% inhibition)
Figure 6. Compound 3 in a concentration-dependent manner inhibits toxicity of
2
3
100
100
100
l
l
l
M (4)
M (4)
M (4)
16.4 4.1
11.2 3.4
24.5 5.2
0.3 2.5
14.2 5.0
14.0 3.3
33.8 19.2
30.8 5.9
THP-1 cell secretions toward SH-SY5Y neuroblastoma cells. THP-1 cells were
pretreated with various concentrations (lM) of inhibitors as described in the legend
4d
Celecoxib
of Figure 4. After 24 h incubation, the cell-free supernatants of THP-1 cells were
transferred to the wells containing SH-SY5Y cells. Viability of SH-SY5Y cells was
assessed after 72 h by the MTT assay (A) and by measuring the LDH activity in the
supernatants (B). Data (means SEM) from 11 independent experiments are
presented. The concentration-dependent effects of compound 3 were assessed by
randomized block design ANOVA.
10 lM (3)
(H2O2) which is often used to model oxidative stress in cell culture
experiments (Fig. 7B).30 Compounds 2 and 4d were ineffective as
neuroprotective agents when added directly to SH-SY5Y cells inde-
pendent of the agent used to induce neuronal death (data not
shown). In the case of compound 3, its direct neuroprotective effect
on SH-SY5Y cells (Fig. 7A) could also be at least partially responsi-
ble for its effect in the anti-neurotoxic assay (Fig. 6B) since some of
the compound could have been transferred to SH-SY5Y cultures
with the supernatants from stimulated THP-1 cells.
2.2.5. Anti-neurotoxic activity of 4d enantiomers
The separate enantiomers of 4d ((+) = 4e and (ꢀ) = 4f) were
each compared with the racemic mixture. Data presented in Figure
8 are comparable to those in Figure 6, except for the fact that THP-
1 cells were incubated in the presence of various compounds and
stimulus for 48 h instead of 24 h. The longer 48 h incubation time
was chosen to achieve more severe neurotoxicity. Longer stimula-
tion period of THP-1 cells indeed resulted in a higher percentage of
SH-SY5Y cell death (cf. Figs. 8 and 6). Also, the anti-neurotoxic ef-
fect of compound 4d was somewhat reduced with the IC50 values
increasing to 14
lM according to the MTT assay ( Fig. 8A) and
40 M according to the LDH measurements (Fig. 8B). Both enanti-
l
omers, 4e and 4f, showed activity similar to the racemic mixture
4d. These results further narrowed the list of candidates for
in vivo experiments to compounds 3, 4d, and its enantiomers 4e
and 4f.
Figure 7. Compound 3 in a concentration-dependent manner protects SH-SY5Y
cells from THP-1 cell secretion-induced toxicity, but not from hydrogen peroxide-
induced lysis. SH-SY5Y cells were seeded into 24-well plates at a concentration of
2 ꢁ 105 cells mLꢀ1 in 0.4 mL of DMEM-F12 medium containing 5% FBS. Various
concentrations of compound 3 were added at the time of transfer of THP-1 cell
We previously reported that NSAIDs (which act by inhibiting
COX) diminished THP-1 cell-induced toxicity towards SH-SY5Y
cells in vitro.28 Here we demonstrate that compounds 2, 3, and
4d–f were reducing neuronal cell killing by THP-1 cells. All five
of these compounds, similarly to the previously studied NSAIDs,
were able to reduce the toxic secretions thus demonstrating anti-
neurotoxic activity on stimulated THP-1 cells. Since these com-
pounds partially inhibit both COX isoforms (Table 2), it is likely
that their in vitro anti-neurotoxic activity is mediated through
the inhibition of prostaglandin production by THP-1 cells.28b Com-
pound 3 had an additional benefit by protecting SH-SY5Y cells
supernatants that had been stimulated with LPS + IFN-
c (A) or culture media
containing 250 M H2O2. Cell death was assessed by the LDH assay 72 h later. Data
l
(means SEM) from five independent experiments are presented. The concentra-
tion-dependent effects of compound 3 were assessed by randomized block design
ANOVA.
THP-1 cells resulted in a concentration-dependent reduction in
neuronal killing according to the LDH assay (Fig. 7A). Compound
3 failed to rescue SH-SY5Y cells killed by hydrogen peroxide

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A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
Table 4
Anti-inflammatory activity of compounds in formalin-induced rat paw edema
bioassay (sub-acute inflammatory model)
Compounda
Volume of edemab (mL)
0
1st day
8th day
Control
3
4d
4e
0.29 0.01
0.31 0.02
0.30 0.01
0.31 0.01
0.28 0.02
0.30 0.01
0.75 0.01
0.83 0.01
0.57 0.01* (43)
0.58 0.01* (39)
0.59 0.01* (39)c
0.55 0.01* (41)
0.59 0.02* (37)
0.61 0.01* (44)
0.63 0.01* (38)
0.67 0.02* (33)
0.63 0.01* (35)
0.67 0.02* (41)
4f
Celecoxib
*
Significantly different compared to respective control values, P < 0.05.
Dose levels of 20 mg/kg (po) were used for all test compounds including
a
celecoxib.
b
Values are expressed as means SEM (N = 5 rats per experimental group).
Values in parentheses (percentage anti-inflammatory activity).
c
Table 5
Anti-inflammatory activity of compounds in the turpentine oil-induced granuloma
pouch model
Compounda
Volume of exudatesb (mL)
Percentage of inhibition
Control
3
4d
4e
2.05 0.03
0.99 0.03*
1.00 0.06*
1.24 0.06*
1.03 0.04*
1.10 0.05*
—
Figure 8. Compounds 4–f show similar anti-neurotoxic effect on THP-1 cell-
mediated killing of SH-SY5Y cells. Experiments were performed as described in the
legend of Figure 6 except that THP-1 cells were incubated in the presence of stimuli
and compounds for 48 h instead of 24 h. Data (means SEM) from 11 independent
experiments are presented. All compounds displayed statistically significant
concentration-dependent effects (P < 0.05) according to the randomized block
design ANOVA.
48
51
39
49
46
4f
Celecoxib
*
Significantly different compared to respective control values, P < 0.05.
Dose levels of 20 mg/kg (po) were used for all test compounds including
a
celecoxib.
from THP-1 cell toxic secretions (Fig. 7A); the biochemical mecha-
nisms responsible for this effect remain to be elucidated.
b
Values are expressed as means SEM (N = 5 rats per experimental group).
2.3. In vivo assays
anti-inflammatory activity was then calculated at 1, 2, 3, and 4 h
after induction. The data are presented in Table 3 as the mean
paw volume (mL) and the percentage anti-inflammatory activity.
All of the test compounds, including celecoxib, significantly re-
duced paw edema volume 1, 2, 3, and 4 h after induction. However,
a comparison of the anti-inflammatory activity of the test com-
pounds, relative to the control, indicated distinctive pharmacoki-
netic profiles. Thus, after 1 h, compounds 4d (50%) and 4e (56%)
showed a potent and more rapid onset of action than celecoxib
(38%). After 2 h, only compound 3 was equally as effective as cele-
coxib in inhibiting the paw edema. After a 3 h time interval com-
pounds 3, 4d, and 4e displayed good anti-inflammatory activity
(38–50%), but none superior to the reference drug. However, after
4 h, the anti-inflammatory activity of compounds 3 (58%), 4d
(50%), and 4e (50%) were equal to or higher than that of celecoxib
(47%).
The anti-inflammatory activity of selected analogs 3, 4d, 4e and
4f were evaluated by two screening protocols, namely, the forma-
lin-induced paw edema and turpentine oil-induced granuloma
pouch bioassays. Celecoxib (20 mg/kg) was used as a reference
standard anti-inflammatory agent. The paw edema was employed
as a model for acute and sub-acute inflammation, while the tur-
pentine oil-induced granuloma pouch assay was utilized as an-
other model for sub-acute inflammatory condition. The data
obtained are presented in Tables 3–5.
2.3.1. Formalin-induced paw edema bioassay
In this acute inflammatory model each test compound was
dosed orally (po) at 20 mg/kg body weight 1 h prior to induction
of inflammation by formalin injection. Celecoxib was utilized as a
reference anti-inflammatory drug at a dose of 20 mg/kg, po. The
Table 3
Anti-inflammatory activity of compounds in formalin-induced rat paw edema bioassay (acute inflammatory model)
Compounda
Volume of edemab (mL)
2 h
0
1 h
3 h
4 h
Control
3
4d
4e
0.29 0.01
0.31 0.02
0.30 0.01
0.31 0.01
0.28 0.02
0.30 0.01
0.45 0.01
0.51 0.01
0.61 0.01
0.67 0.01
0.40 0.04* (44)c
0.38 0.01* (50)
0.38 0.01* (56)
0.38 0.03* (37)
0.40 0.02* (38)
0.44 0.01* (41)
0.46 0.01* (27)
0.45 0.01* (36)
0.43 0.02* (32)
0.43 0.02* (41)
0.47 0.01* (50)
0.47 0.02* (46)
0.49 0.01* (44)
0.48 0.02* (38)
0.46 0.01* (56)
0.47 0.01* (58)
0.49 0.01* (50)
0.50 0.01* (50)
0.51 0.02* (39)
0.50 0.01* (47)
4f
Celecoxib
*
Significantly different compared to respective control values, P < 0.05.
a
Dose levels of 20 mg/kg (po) were used for all test compounds including celecoxib.
Values are expressed as means SEM (N = 5 rats per experimental group).
b
c
Values in parentheses represent percentage anti-inflammatory activity.

A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
5691
Table 6
granuloma inhibition was calculated. Celecoxib (20 mg/kg) was
used as a reference drug. Table 5 illustrates that all of the studied
compounds significantly reduced the exudate volume when com-
pared to the control group of animals. Compounds 4d, 4f, and 3 ap-
peared to be more potent than celecoxib (46%) with percentages of
granuloma inhibition of 51%, 49%, and 48%, respectively.
The in vivo experiments (Tables 3–5) demonstrated anti-
inflammatory activity of compounds 3 and 4d in formalin paw ede-
ma model of acute inflammation, as well as in the formalin paw
edema and turpentine oil-induced granuloma pouch assays (sub-
acute inflammatory models). Therefore these compounds might
be effective in managing acute inflammation, as well as in control-
ling chronic inflammatory conditions.
The anti-inflammatory activity (ED50), ulcerogenic effects, and acute LD50 of test
compounds
Test compound
ED50 (mg/kg)
% Ulceration
ALD50 (mg/kg)
Indomethacin
3
4d
4e
—
100
0.0
10.0
0.0
0.0
0.0
—
17.50
19.64
18.95
22.41
15.65
>300
>300
>300
>300
—
4f
Celecoxib
2.3.2. Formalin-induced paw edema bioassay (sub-acute
inflammatory model)
In this sub-acute inflammatory model, inflammation was in-
duced by formalin injection on the first and third days, and test
compounds were administered orally (at 20 mg/kg daily) for
7 days. Again, celecoxib was used as a reference anti-inflammatory
agent in this assay. The anti-inflammatory activity was calculated
at the 1st and 8th day after induction and the data presented in
Table 4 as the mean paw volume and the percentage anti-inflam-
matory activity relative to a control.
The obtained data revealed that, according to the measure-
ments performed on both the 1st and 8th day, all tested com-
pounds displayed significant anti-inflammatory activity. Percent
inhibition on the 1st day was the highest for compound 3 (43%),
which was greater than celecoxib (37%). At the 8th day, compound
3 was found to have an anti-inflammatory activity higher than the
reference drug with a percentage inhibition of 44% while com-
pound 4d was nearly effective (38%) as celecoxib (41%).
2.3.4. Effective dose 50 (ED50) in formalin-induced paw edema
bioassay
The tested compounds were further evaluated for their ED50 in
male rats 3 h after formalin injection. The results indicated that
compounds 3, 4d, and 4e have ED50 similar to celecoxib suggesting
that they were nearly equipotent to the reference drug.
2.3.5. Ulcerogenic activity
The tested compounds that exhibited variable anti-inflamma-
tory profiles were further evaluated for their ulcerogenic potential
in rats. Gross observation of the isolated rat stomachs showed a
normal stomach texture for all of the tested compounds with no
observable hyperemia. This indicated a superior GI safety profile
(0–10% ulceration) in the population of the fasted rats. It is note-
worthy that, indomethacin—the reference standard anti-inflamma-
tory drug—caused 100% ulceration under the same experimental
conditions (Table 6).
2.3.3. Turpentine oil-induced granuloma pouch bioassay
In this sub-acute inflammatory model, each test compound was
administered orally (20 mg/kg) 1 h prior to turpentine oil injection
and administration was continued for 7 days. At the 8th day, the
volume of exudates (mL) was measured and the percentage of
2.3.6. Acute toxicity
All of the selected compounds were further evaluated for their
approximate acute lethal dose (ALD50) in male rats. All of the
Figure 9. COX-2 superimposition of the co-crystallized celecoxib derivative SC-558 (colored red) and the docked compound 4e (colored green). Pink dashed lines depict
hydrogen bond interactions. Viewed using MOE module.

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A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
Figure 10. COX-2 superimposition of the co-crystallized celecoxib derivative SC-558 (colored red) and the docked compound 4f (colored green). Pink dashed lines depict
hydrogen bond interactions. Viewed using MOE module.
tested compounds proved to be non-toxic and well-tolerated by
the experimental animals. The compounds showed a high safety
margin when screened at graded doses (10–300 mg/kg, po), where
their ALD50 values were found to be >300 mg/kg (Table 6).
nyl moiety of SC-558 and is surrounded by hydrophobic residues
Tyr355, Ph518, and Val523. The docking study shows that the
enantiomeric compounds 4e and 4f have a binding pattern in the
COX-2 site which is close to the pattern observed in the crystal
structure of the SC-558 complex with comparable docking scores.
This may account for the in vitro COX-2 inhibition recorded for
the racemic mixture (Table 2). These observations may also explain
the very similar anti-neurotoxic activity of the two enantiomers
(Fig. 8).
3. Docking study
The inhibition of the COX-2 enzyme by compound 4d (Table 2)
encouraged us to investigate the structural basis underlying the
docking of both enantiomers 4e and 4f. The docking study was
carried out using the enzyme parameters obtained from the crys-
tallographic structure of the complex between COX-2 with the
co-crystallized celecoxib derivative SC-558 (PDB ID: 1CX2).31 The
docking simulation for the ligands was carried out using molecular
operating environment (MOE) software supplied by the Chemical
Computing Group, Inc., Montréal Canada.32
In the SC-558 molecule, hydrogen bonding is shown between
the oxygen atom and the imidazole NH His90, and also between
the sulfonamide NH2 group of SC-558, the carbonyl oxygen of
Gln192 and carbonyl oxygen of Leu35. Whereas in compound 4e,
hydrogen bonding is observed between the nitrogen of the cyano
group and hydroxyl group of Ser530 as well as between the car-
bonyl oxygen of compound 4e with the NH of Arg120 (Fig. 9).
Figure 10 shows COX-2 superimposition of SC-558 and the
docked (ꢀ)-enantiomer 4f. In this case, hydrogen bonding is ob-
served between the cyano group nitrogen and both the hydroxyl
groups of Tyr385 and Ser530. Hydrogen bonding is also seen be-
tween the carbonyl oxygen of compound 4f and the hydroxyl
group of Ser530.
4. Conclusion
A novel dipyrazole ethandiamide lead compound 3 has been
synthesized and derivatized through the nucleophilic displace-
ment of only one pyrazole group. The resultant novel ethandia-
mide compounds have been investigated for possible anti-
inflammatory and neuroprotective properties using human cell
lines as surrogates of glial and neuronal cells. In vitro cell culture
techniques and enzymatic assays were utilized to screen the com-
pounds for their ability to affect a number of biological properties
including: (1) cell viability; (2) secretion of a pro-inflammatory
chemokine; (3) the COX enzymatic activity and (4) monocytic
cell-induced killing of neuronal cells. The synthesized compounds
were also evaluated for their in vivo anti-inflammatory activity
using two different screening protocols, namely, formalin-induced
paw edema and the turpentine oil-induced granuloma pouch
bioassays.
Among the tested analogs, compounds 2, 3, and 4d showed
in vitro anti-neurotoxic and neuroprotective activity at concentra-
tions below their toxic range and partially inhibited both COX-1
and COX-2 enzymes. It should be noted that both enantiomers
4e and 4f showed in vitro activity as anti-neurotoxic agents very
similar to the racemic mixture 4d. Comparable docking scores with
the COX-2 enzyme active site also suggested that 4e and 4f may
In Figures 9 and 10, the methyl benzyl group of enantiomers 4e
and 4f adopt the same position as the trifluoromethyl group of SC-
558 and is bound in a hydrophobic cavity formed by Met113,
Val116, Val349, and Leu359. In addition, the N-phenyl ring of com-
pounds 4e and 4f adopts the same position as the p-sulfamoylphe-

A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
5693
possess similar anti-inflammatory properties in line with the
in vitro results. Compounds 3 and 4d were notable for their more
potent and faster onset of action than the clinically available
COX-2 inhibitor celecoxib in both the acute inflammatory and
sub-acute inflammatory models. This suggests that they might be
effective in managing acute inflammation, as well as in controlling
chronic inflammatory conditions. Additionally, all of the selected
compounds revealed excellent GI safety profile and were well-tol-
erated by experimental animals with a high safety margin (ALD50
>300 mg/kg). Finally, compound 4d was identified as a novel neu-
roprotective and anti-inflammatory agent within this study. Com-
pounds 3 and 4d were the most efficacious derivatives and
represented sterically smaller and more lipophilic molecules in
comparison to the other derivatives. These attributes may have
facilitated their passage through cell membranes and accounted
for their activity. Future work will attempt to confirm this hypoth-
esis through a systematic and rigorous battery of derivatizations of
various ethandiamide parent compounds. The pyrazole ethandia-
mide compounds represent a fruitful matrix for the future develop-
ment of a new class of neuroprotective and anti-inflammatory
agents.
an 84% yield; mp 160–162 °C; IR (cmꢀ1): 1735 (CO), 1692 (CO
amide); 1H NMR (d ppm): 12.85 (s, 1H, NH, D2O exchangeable),
8.42 (s, 1H, pyrazole-C5–H), 8.02–7.42 (m, 5H, aromatic-H), 3.93
(s, 1H, CH3); 13C NMR (d ppm): 160.1, 157.1, 150.8, 146.3, 137.9,
136.3, 129.4, 127.6, 122.1, 108.5, 53.7. Calculated for
(C13H11N4O3 = M+1) 271.0831, found 271.0811.
5.1.2. N1,N2-Bis(4-cyano-1-phenyl-1H-pyrazol-5-yl)oxalamide
(3)
To a stirred solution of 5-amino-1-phenyl-1H-pyrazole-4-car-
bonitrile 1 (1.84 g, 0.01 mol) in tetrahydrofuran (THF) (10 mL)
was added oxalyl chloride (1.27 g, 0.01 mol) dropwise over a peri-
od of 30 min. The reaction mixture was stirred for further 24 h at
room temperature (rt). The solvent was evaporated under reduced
pressure, diethyl ether is then added and the product was filtered
and recrystallized from methanol (yield 85%); mp 262–264 °C; IR
(cmꢀ1): 3218 (NH), 2237 (CN), 1689 (CO amide); 1H NMR (d
ppm): 11.78 (s, 1H, NH, D2O exchangeable), 8.35 (s, 1H, pyrazole-
C5–H), 7.53–7.46 (m, 5H, aromatic-H); 13C NMR (d ppm): 157.8,
142.7, 139.3, 137.3, 129.5, 129.1, 123.8, 112.4, 90.5. Calculated
for (C22H15N8O2 = M+1) 423.1318, found 423.1357.
5.1.3. 2-Oxo-2-substituted-N-[4-cyano-1-phenyl-1H-pyrazol-5-
yl] acetamides (4)
5. Experimental
5.1. Chemistry
A mixture of oxalamide derivative 4 (0.42 g, 0.001 mol) and the
appropriate amine (0.004 mol) in dry THF (10 mL) was heated un-
der reflux for 2 h. The solvent was evaporated under reduced pres-
sure, diethyl ether added and the formed product was filtered,
washed with diethyl ether, dried and crystallized from chloro-
form–hexane.
Unless otherwise specified, all reagents were used as supplied
by either the Sigma–Aldrich-Fluka (St. Louis, MO, USA) or Fisher
Scientific (Ottawa, ON, Canada) chemical companies. Reaction sol-
vents were dried by storage over 3 Å molecular sieves. Melting
points were determined in open glass capillaries on a Stuart melt-
ing point apparatus and are uncorrected. IR spectra in cmꢀ1 were
recorded on a FTIR Nicolet IR 200 spectrophotometer. 1H NMR
and 13C NMR spectra were run on an AS 400 MHz NMR Oxford
Spectrophotometer, using tetramethylsilane (TMS) as the internal
standard and DMSO-d6 as the solvent. Chemical shifts were re-
corded as d (ppm). Mass spectrometry data was collected by time
of flight mass spectrometry (Micromass LCT Premier TM series
(ToF) MS; Waters Inc.). The system was set up to acquire in positive
electrospray ionization (ESI+) in W mode. The following source
parameters were used for instrumental analysis: capillary voltage
(2900 V), sample cone voltage (30 V), desolvation temperature
(350 °C), source temperature (120 °C), gas flow through the cone
(20 mL/min), and desolvation gas flow (750 mL/min). The optical
parameters of the instrument were as follows: aperture 1 voltage
(15 V), ion energy (35 V), aperture 2 voltage (4.0 V), hexapole volt-
age (5.0 V), attenuated Z focus voltage (250 V), ToF flight tube volt-
age (7200 V), reflection voltage (1800 V), pusher (940 V), puller
voltage (820 V), and MCP detector voltage (2050 V). Purity of all
samples was determined using elemental analyses with an EA
1108 elemental analyzer. Reactions were monitored by TLC analy-
sis using Merck Silica Gel 60 F-254 thin layer plates. Flash chroma-
tography was carried out on silica gel (230–400 mesh). All
compounds showed purities equal to or greater than 95% as deter-
mined by elemental analysis.
5.1.3.1. 2-Oxo-2-phenethylamino-N-[4-cyano-1-phenyl-1H-pyr-
azol-5-yl] acetamide (4a). Yield 80%; mp 168–170 °C; IR (cmꢀ1):
3229 (NH), 2232 (CN); 1H NMR (d ppm): 9.13 (s, 1H, NH, D2O
exchangeable), 8.33 (s, 1H, pyrazole-C5–H), 7.54–7.15 (m, 10H, aro-
matic-H), 3.36 (q, 2H, J = 8 HZ, CH2), 2.78 (t, 2H, J = 8 Hz, CH2); 13C
NMR (d ppm): 159.7, 157.9, 142.5, 138.9, 137.2, 129.4, 128.9, 128.6,
128.3, 126.2, 123.8, 112.4, 90.4, 40.6, 34.3. Calculated for
(C20H18N5O2 = M+1) 360.1461, found 360.1453.
5.1.3.2. 2-Morpholinopropyl 2-oxo-N-[4-cyano-1-phenyl-1H-
pyrazol-5-yl] acetamide (4b). Yield 82%; mp 138–140 °C; IR
(cmꢀ1): 3227 (NH), 2234 (CN); 1H NMR (d ppm): 9.72 (s, 1H, NH,
D2O exchangeable), 8.94 (s, 1H, pyrazole-C5–H), 8.53–8.23 (m,
5H, aromatic-H), 4.07 (t, 2H, J = 6 Hz, CH2), 3.37 (t, 2H, J = 6 Hz,
CH2), 2.49–2.47 (m, 6H, CH2); 13C NMR (d ppm): 159.7, 157.9,
142.1, 138.5, 128.9, 127.5, 123.3, 114.2, 90.4, 66.1, 56.2, 53.3,
40.1, 25.3. Calculated for (C19H23N6O3 = M+1) 383.1831, found
383.1838.
5.1.3.3. 2-Cyclohexylamino-2-oxo-N-[4-cyano-1-phenyl-1H-pyr-
azol-5-yl] acetamide (4c). Yield 82%; 212–213 °C; IR (cmꢀ1):
3229 (NH), 2234 (CN); 1H NMR (d ppm): 8.99 (s, 1H, NH, D2O
exchangeable), 8.30 (s, 1H, pyrazole-C5–H), 7.7–7.3 (m, 5H, aro-
matic-H), 1.7–1.2 (m, 10H, CH2); 13C NMR (d ppm): 159.9, 157.2,
142.5, 137.3, 129.4, 128.9, 123.9, 112.5, 90.2, 48.7, 31.6, 24.7. Cal-
culated for (C18H20N5O2 = M+1) 338.1617, found 338.1585.
5.1.1. Methyl 4,5-dihydro-4-oxo-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine-6-carboxylate (2)
5.1.3.4. R/S-( )-2-Methylbenzylamino 2-oxo-N-[4-cyano-1-phe-
nyl-1H-pyrazol-5-yl] acetamide (4d). Yield 85%; mp 180–182 °C;
IR (cmꢀ1): 3229 (NH), 2220 (CN); 1H NMR (d ppm): 8.07 (s, 1H, NH,
D2O exchangeable), 7.70 (s, 1H, pyrazole-C5–H), 7.7–7.35 (m, 10H,
aromatic-H), 4.35 (q, 1H, J = 7.2 Hz, CH), 1.44 (d, 3H, J = 7.2 Hz,
CH3); 13C NMR (d ppm): 161.9, 148.1, 142.1, 139.6, 138.5, 128.9,
128.7, 128.4, 127.4, 126.7, 123.3, 114.5, 86.2, 50.0, 20.9. Calculated
for (C20H18N5O2 = M+1) 360.1460, found 360.1461.
To a stirred solution of oxalyl chloride (1.27 g, 0.01 mol) in THF
(5 mL), a solution of 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile
1 (0.92 g, 0.005 mol) in THF (5 mL) was added dropwise over a per-
iod of 2 h. The reaction mixture was further stirred for 24 h at rt.
The solvent was evaporated under reduced pressure, and the
formed product was filtered, dried and refluxed in excess methanol
for 5 min. The beige crystals were collected by suction filtration in

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A. M. Youssef et al. / Bioorg. Med. Chem. 18 (2010) 5685–5696
5.1.3.5. R-(+)-2-Methylbenzylamino 2-oxo-N-[4-cyano-1-phenyl-
1H-pyrazol-5-yl] acetamide (4e). Yield 85%; mp 180–182 °C; IR,
1H NMR, 13C NMR same as above. Calculated for (C20H18N5O2 =
M+1) 360.1460, found 360.1457.
directly to the SH-SY5Y cells at the time of transfer of media. Cell
death was assessed by the LDH assay 72 h later.
5.2.6. Cell viability assays: LDH release
Cell death was evaluated by LDH release. LDH activity in cell
culture supernatants was measured by an enzymatic test in which
formation of the formazan product of iodonitrotetrazolium dye
5.1.3.6. S-(ꢀ)-2-Methylbenzylamino 2-oxo-N-[4-cyano-1-phenyl-
1H-pyrazol-5-yl] acetamide (4f). Yield 83%; mp 180–182 °C; IR,
1H NMR, 13C NMR same as above. Calculated for (C20H18N5O2 =
M+1) 360.1460, found 360.1449.
was followed colorimetrically.33 Briefly, 100
l
L of cell culture
supernatants were pipetted into the wells of 96-well plates, fol-
lowed by addition of 15
L lactate solution (36 mg mLꢀ1) and
15
L p-iodonitrotetrazolium violet solution (2 mg mLꢀ1). The
l
5.2. In vitro assays
l
enzymatic reaction was started by addition of 15 l
L of NAD+/
5.2.1. In vitro reagents
diaphorase solution (3 mg mLꢀ1 NAD+; 2.3 mg solid mLꢀ1 diapho-
rase). After 15–30 min incubation, the reaction was terminated
The following substances used in various assays were obtained
from Sigma: hydrogen peroxide, bacterial lipopolysaccharide (LPS,
from Escherichia coli 055:B5), diaphorase (EC 1.8.1.4, from Clostrid-
ium kluyveri, 5.8 U mgꢀ1 solid), DMSO, p-iodonitrotetrazolium
violet, NAD+, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
by addition of 15 l
L oxamate (16.6 mg mLꢀ1). Optical densities at
490 nm were measured by a microplate reader, and the amount
of LDH which had been released was expressed as a fraction of
the value obtained in comparative wells where the remaining cells
were totally lysed by 1% Triton X-100.
zolium bromide). Human recombinant IFN-c, MCP-1 and antibodies
used in MCP-1 enzyme linked immunoabsorbent assay (ELISA) were
purchased from Peprotech (Rocky Hill, NJ, USA).
5.2.7. Cell viability assays: reduction of formazan dye (MTT)
The MTT assay was performed using a previously described
method,34 which is based on the ability of viable, but not dead
cells, to convert the tetrazolium salt (MTT) to colored formazan.
The viability of SH-SY5Y cells was determined by adding MTT to
5.2.2. Cell culture
The human monocytic THP-1 cell line was obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). The
human neuroblastoma SH-SY5Y cell line was a gift from Dr. R. Ross,
Fordham University, NY. These cells were grown in Dulbecco’s
modified Eagle’s medium-nutrient mixture F12 ham (DMEM-F12)
supplemented with 10% fetal bovine serum (FBS) supplied by Ther-
mo Scientific HyClone (Logan, UT, USA). Both cell lines were used
without initial differentiation.
the SH-SY5Y cell cultures to reach
a final concentration of
0.5 mg mLꢀ1. Following a 1 h incubation at 37 °C, the dark crystals
formed were dissolved by adding to the wells an equal volume of
SDS/DMF extraction buffer (20% sodium dodecyl sulfate, 50% N,N
dimethyl formamide, pH 4.7). Subsequently, plates were placed
overnight at 37 °C; optical densities at 570 nm were then mea-
sured by transferring 100 lL aliquots to 96-well plates and using
5.2.3. Effects of compounds on THP-1 cells viability and MCP-1
secretion
a platereader. The viable cell value was calculated as a fraction of
the value obtained from cells incubated with fresh medium only.
Human monocytic THP-1 cells were seeded into 24-well plates
at a concentration of 5 ꢁ 105 cells mLꢀ1 in 0.9 mL of DMEM-F12
medium containing 5% FBS. The cells were incubated in the pres-
ence or absence of various compounds or their vehicle solution
(DMSO) for 15 min prior to the addition of an activating stimulus
5.2.8. Cyclooxygenase (COX) enzymatic assay
Compounds were tested for their ability to inhibit the two COX
isoforms by using COX inhibitor screening assay kit supplied by the
Cayman Chemical Company (Ann Arbor, MI, USA) according to the
protocols provided by the manufacturer. This kit includes ovine
COX-1 and human recombinant COX-2 enzymes, and it measures
percent inhibition of prostaglandin production by the various com-
pounds for the two COX isoforms.
(0.5
100
l
g mLꢀ1 LPS with 150 U mLꢀ1 IFN-
c
). After 24 h incubation,
lL of THP-1 culture media were sampled for lactate dehydro-
genase (LDH) to determine percentage of dead cells, while evalua-
tion of surviving cells was performed by the MTT assay. In addition,
concentration of MCP-1 (ng mLꢀ1) was measured in 100
lL of cell-
free culture medium by ELISA according to the protocol provided
5.3. In vivo anti-inflammatory activity
by the supplier of the antibodies (Peprotech).
5.3.1. Animal experiments
5.2.4. Cytotoxicity of THP-1 cells toward SH-SY5Y
neuroblastoma cells
Male Wistar strain albino rats weighing 180–200 g were used
throughout the assay. They were acquired from a closed random
bred colony at the Faculty of Veterinary Medicine, University of
Alexandria, Egypt. Rats were given ad libitum access to food and
water and housed in groups of four in isolated cages under stan-
dard conditions of light and temperature. The animals were accli-
matized for 2 weeks prior to experiments. The investigation
conformed to the guide for the Care and Use of Laboratory Animals
published by US National Institute of Health (NIH Publication No.
83-23, revised 1996). The Local Ethics Committee approved this
study.
THP-1 cells were seeded into 24-well plates and stimulated in
the presence and absence of various compounds as described
above. After 24 or 48 h incubation, 0.4 mL of cell-free supernatant
was transferred to each well containing SH-SY5Y cells. The cells
had been plated 24 h earlier at a concentration of 2 ꢁ 105 cells
mLꢀ1 in 0.4 mL of DMEM-F12 medium containing 5% FBS. After
72 h of incubation, the neuronal culture media were sampled for
LDH to determine release from dead cells, while evaluation of sur-
viving cells was performed by the MTT assay.
5.2.5. Neuroprotective effects
5.3.2. Formalin-induced paw edema bioassay
SH-SY5Y cells were seeded into 24-well plates at a concentra-
tion of 2 ꢁ 105 mLꢀ1 in 0.4 mL of DMEM-F12 medium containing
5% FBS. After 24 h incubation cell culture medium was replaced
by either supernatants form THP-1 cells that had been stimulated
This acute inflammatory model was performed as previously
described.35 The rats were randomly divided into groups of five with
one group as a control. The other groups received the standard drug
celecoxib or the test compounds (at a dose of 20 mg/kg body weight
po). A solution of formalin (2%, 0.1 mL) was injected into the sub-
planter region of the left hind paw under light ether anesthesia 1 h
for 24 h with LPS + IFN-
c or culture media containing 250 lM
H2O2. Various concentrations of the compounds were added

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5695
after oral administration (po) of the test compound (at a dose level of
5.3.8. Statistical analysis
20 mg/kg body weight). The paw volume (mL) was measured by
means of water plethysmometer and re-measured again 1, 2, 3,
and 4 h after administration of formalin. The edema was expressed
as an increase in the volume of paw, and the percentage of edema
inhibition for each rat and each group was obtained as follows:
Data are presented as means standard error of the mean
(SEM). The concentration-dependent effects of various drugs
in vitro were evaluated statistically by the randomized block de-
sign analysis of variance (ANOVA). Data obtained in animal studies
were subjected to ANOVA, followed by Student–Newman–Keuls
Multiple Comparison Test. The difference in results was considered
significant when P < 0.05.
% Inhibition ¼ ððVt ꢀ VoÞcontrol
ꢀ ðVt ꢀ VoÞtested compoundÞ=ðVt ꢀ VoÞcontrol ꢁ 100
6. Modeling studies
where Vt is the volume of edema at specific time interval, Vo is the
Computer-assisted simulated docking experiments were car-
ried out under an MMFF94X force field in COX-2 structure (PDB
ID: 1CX2) using Chemical Computing Group’s Molecular Operating
Environment (MOE-dock 2008) software, Montréal, Canada.
volume of edema at zero time interval.
5.3.3. Formalin-induced paw edema bioassay
This sub-acute inflammatory model was performed as previ-
ously described.35 Rats in the first experiment were given the same
test compounds at a dose level of 20 mg/kg body weight daily for 7
consecutive days. A solution of formalin (2%, 0.1 mL) was injected
into the subplanter region of the left hind paw under light ether
anesthesia 1 h after oral administration (po) of the test compound.
A second injection of formalin (2%, 0.1 mL) was given on the third
day. The changes in the volume of paw were measured plethysmo-
graphically at the first and eighth days.
Supplementary data
Instrumental data of some compounds is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgments
The authors gratefully acknowledge financial support provided
by grants from the Natural Sciences and Engineering Research
Council of Canada, the Jack Brown and Family Alzheimer’s Disease
Research Foundation, and the I.K. Barber School of Arts and Sci-
ences, UBC Okanagan. The authors also thank Dr. Patrick Shipman
for valuable discussions and instrumental help.
5.3.4. Turpentine oil-induced granuloma pouch bioassay
This sub-acute inflammatory model was performed as previ-
ously described.35b,36 A subcutaneous dorsal granuloma pouch
was made in ether-anesthetized rats by injecting 2 mL of air, fol-
lowed by injection of 0.5 mL of turpentine oil into it. All of the test
compounds were administered orally (at a dose level of 20 mg/kg
body weight) one h prior to turpentine oil injection and continued
for seven consecutive days. On the eighth day, the paw was opened
under anesthesia and the exudates were taken out with a syringe.
The volume (mL) of the exudates was measured and the percent-
age inhibition of inflammation relative to the control was deter-
mined as follows:
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